CN112695128A - Rapid analysis method for yeast fungi based on membrane culture - Google Patents
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Abstract
The invention discloses a rapid analysis method of yeast type fungi based on membrane culture, which comprises the following steps of S1, experimental materials (1) candida albicans standard strain, candida tropicalis standard strain, candida krusei standard strain, candida glabrata standard strain, 30 candida albicans clinical isolates, 21 candida tropicalis clinical isolates, 15 candida krusei clinical isolates and 12 candida glabrata clinical isolates; s2, culturing the strains, wherein according to different experimental purposes, all the strains in the experiment are cultured in a Sapaul solid culture medium (prepared in a laboratory) at 30 ℃ or in a yeast extract-peptone-glucose (YPD) culture solution (prepared in the laboratory) at 30 ℃ and under 220rpm shaking. The rapid analysis method of the yeast fungi based on the membrane culture can not only retain the advantages of the culture method to achieve enrichment and enrichment treatment of fungi samples, but also can be linked with high-sensitivity qPCR, so that the method only needs to obtain the sufficient quantity of the fungi for qPCR detection, and does not need long-time culture.
Description
Technical Field
The invention relates to the technical field of fungus analysis research, in particular to a rapid analysis method of yeast-type fungi based on membrane culture.
Background
The fungal infection mainly aims at people with low immunity, the arrival of an aging society, the frequent occurrence of AIDS, diabetes, malignant tumor, blood disease and autoimmune disease and the wide application of various medicaments, the fungal infection rate, the incidence rate of the fungal disease and the mortality rate are on the rising trend year by year, in China, the fungal infection accounts for 10-20% of the total number of various pathogenic bacteria, the incidence rate of the fungal infection is increased by 3 times in the United states within 20 years, the pathogens of the fungal infection are more than candida, and causes the highest mortality rate caused by deep infection such as blood system infection, fungal meningitis and the like, clinically common candida pathogens comprise candida albicans, candida tropicalis, candida glabrata, candida krusei and the like, the proportion of candida albicans is the largest, the structures of fungi and bacteria are different, and the selection of medicines in antifungal treatment needs to be careful.
The existing fungal infection diagnosis method has the characteristics of less methods, slow detection and low precision, the culture method is time-consuming, and the detection sensitivity is also influenced by the quantity of bacteria in a sample; beta-D-glucan immunological detection species are poor in specificity, and in addition, Candida krusei and Candida glabrata have natural drug resistance to main antifungal drugs, namely fluconazole and itraconazole, so that a rapid and accurate fungus identification method is very necessary to be researched and developed.
In order to solve the above problems, there is an urgent need for analyzing and researching fungi by a rapid analysis method of yeast-type fungi based on membrane culture.
Disclosure of Invention
The invention aims to provide a rapid analysis method of yeast fungi based on membrane culture, which aims to solve the problems that the culture method proposed in the background art is time-consuming, the beta-D-glucan immunology detection species specificity is poor, and in addition, candida krusei and candida glabrata have natural drug resistance to main antifungal drugs of fluconazole and itraconazole and are not beneficial to rapid and accurate identification and analysis of fungi.
In order to achieve the purpose, the invention provides the following technical scheme: a rapid analysis method of yeast-type fungi based on membrane culture comprises the following steps:
s1 test Material
(1) Candida albicans standard strain, Candida tropicalis standard strain, Candida krusei standard strain, Candida glabrata standard strain, 30 Candida albicans clinical isolates, 21 Candida tropicalis clinical isolates, 15 Candida krusei clinical isolates and 12 Candida glabrata clinical isolates.
(2) All clinical isolates used in this experiment have been identified as species using a Candida chromogenic medium.
S2 strain culture
According to different purposes, all strains in the experiment are cultured in a Sapaul solid culture medium (prepared in a laboratory) at 30 ℃ or in a yeast extract peptone glucose (YPD) culture solution (prepared in the laboratory) at 30 ℃ and under 220rpm shaking.
S3 Candida primer selection
Detection primers were selected at different levels from the ribosomal DNA gene coding regions of Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei, namely the 18SrRNA, the transcribed spacer-1, 5.8SrRNA, the transcribed spacer-2 and 28SrRNA five segments, namely the fungal universal primer ITS84/ITS6, the Candida universal primer pCps-1F/1R, the Candida albicans specific primer Caps-1F/1R, the Candida tropicalis specific primer CTR1/2, the Candida glabrata specific primer CGL1/2 and the Candida krusei specific primer CKRU 1/2.
S4, Membrane culture
(1) The culture medium used in the membrane culture technique comprises two parts, a Sapaul plate with a white microporous filter membrane paved on the surface and a chromogenic plate without nutrient components, wherein the cellulose microporous filter membrane allows water and nutrient exchange, but prevents candida cells from passing through, and the chromogenic plate is made of 1% agarose gel containing 0.8mg/ml thiazole blue.
(2) Selecting a Candida albicans colony under aseptic condition, diluting the Candida albicans colony with YPD culture solution 1:1000, respectively taking 10 mu l of diluted bacterial solution, coating an inoculation membrane plate, culturing at 30 ℃, respectively taking out filter membranes at 2h, 4h, 6h, 8h, 12h and 24h, transferring the filter membranes to a color development plate, simultaneously observing the color development condition of the colony on the filter membranes and the growth condition of the colony cultured conventionally, and setting three parallel at each time point.
S5 colony PCR
(1) And (3) selecting the candida tenuis colonies which are subjected to membrane culture and have color development indication, suspending the candida tenuis colonies in 20 mu l of candida lysate, carrying out vortex oscillation, incubating for 5min at 98 ℃ after short vortex oscillation, centrifuging for 10min at 1500Xg, collecting supernatant, diluting with sterile ultrapure water according to a ratio of 1: 5-1: 20, and then respectively using the supernatant as a template to carry out PCR test.
(2) Candida albicans specific primers Casps-1F/1R are selected, the annealing temperature is 58 ℃, sterile ultrapure water is used as a template to serve as a blank control, and the amplification result is analyzed through 2% agarose gel electrophoresis.
S6 extraction of Candida genomic DNA
Respectively taking 1ml of fresh candida bacterial liquid, placing the fresh candida bacterial liquid in a 1.5ml sterile centrifuge tube, centrifuging for 1min at 13400 Xg, discarding supernatant, carrying out wall breaking, column combination, rinsing, elution and other treatments on candida thalli by using a yeast genome DNA extraction kit, finally obtaining a genome sample, quantifying by using an ultraviolet spectrophotometer, and freezing and storing at-30 ℃ for later use.
S7, conventional PCR analysis of primer specificity and detection Limit
(1) And (3) respectively taking 1ng of four candida genomic DNAs, common bacterial genomic DNAs and human genomic DNAs as templates, testing the specificity of the selected 6 pairs of primers according to the PCR parameter setting of the step S5, and taking sterile ultrapure water as a template as a blank control.
(2) Analyzing the amplification result by agarose gel electrophoresis, and respectively carrying out 10-10 ng on 1ng of four candida genomic DNAs6Diluting by times, testing the conventional PCR detection limit of 6 pairs of primers, taking sterile ultrapure water as a template as a blank control, and analyzing the amplification result by agarose gel electrophoresis.
S8 qPCR detection and melting curve analysis of each primer specificity and detection limit
(1) qPCR analysis is carried out on the specificity and detection limit of each primer in the step S7 by adopting an iQ5qPCR instrument, and SYBRGreen Premix Ex is utilizedTaqThe kits were prepared with 25. mu.l each.
(2) The qPCR program was set to 95 ℃ pre-denaturation for 30s, "95 ℃, 5s → 58 ℃, 5s → 72 ℃, 5 s" repeated 35 cycles.
(3) Melting curve analysis was then performed: the temperature was raised from 60 ℃ to 95 ℃ at a rate of 0.1 ℃/s, the change in fluorescence intensity of each qPCR amplicon was recorded, a melting curve was generated, each qPCR threshold was determined by the iQ5 software system default, sterile ultrapure water was used as a template as a blank control, and a set of replicates was set.
S9, qPCR verification of clinical isolate
And (3) carrying out qPCR detection and melting curve analysis on 78 clinical isolates of the candida, and respectively carrying out amplification of a plurality of target points on each candida by using a fungus universal primer, a candida universal primer and a candida specific primer.
S10, data analysis
Mean standard deviation analysis was performed on qPCR data using iQ5 analysis software and microsoft Excel tool.
S11, result
(1) Results of membrane culture
Compared with the conventional culture method, the culture time can be shortened by more than 50%, as shown in figure 1-2, the blue-violet colony spots can be seen by naked eyes after the filter membrane cultured for 8h is developed.
(2) Colony PCR results
The results show that the colony is stained by thiazole blue, the PCR is not affected, except that the PCR of lysate of the colony at a ratio of 1:5 is negative, the templates of the other three dilutions are amplified to form a single band, the size of the product is consistent with the expected result of 272bp, the PCR negative is probably caused by excessively high concentration of the lysate, and the results are shown in FIG. 3:
lane M: DNAmarker;
lane 1: diluting bacterial colony lysate by 1: 5;
lane 2: diluting bacterial colony lysate by 1: 10;
lane 3: diluting bacterial colony lysate at 1: 15;
lane 4: diluting bacterial colony lysate by 1: 20;
lane 5: blank control.
(3) Detection limits and specificities of conventional PCR and qPCR
1. Compared with the detection limit of each primer in the conventional PCR of 1 pg-100 pg, the detection limit of each primer in the qPCR is 1 fg-100 fg, and the overall sensitivity is 1000 times higher than that of the conventional PCR, as shown in FIGS. 4-9 (the abscissa of the qPCR melting curve is temperature (DEG C), and the ordinate is melting peak), the negative derivative (-dRFU/dT) of the relative fluorescence unit data of the amplified gene at each temperature shows a falling trend along with the reduction of the template amount, and the detection limit is the minimum template DNA amount capable of obtaining the melting peak.
2. After the specificity of each primer is analyzed by the electrophoresis of the conventional PCR amplification product, the band specificity is single, but the resolution of agarose electrophoresis is not high, bands with approximate sizes can not be further identified, and qPCR combined melting curve analysis results show that after each candida is identified by three target points qPCR, the triple melting temperatures of the four candida species are 86.5-85.5 ℃ of white, 74.5-85 ℃ of tropical, 74.5-85 ℃ of smooth, 74.5-84.5-75 ℃ of smooth, 75-86.5-90 ℃ of krusei respectively according to the sequences of the fungal universal-candida specific primers, and the results are shown in fig. 12-15 (in the figures, A-D are the triple melting peaks of candida albicans, candida tropicalis, candida glabrata and candida krusei respectively).
(4) Clinical isolates of Candida qPCR analysis
As shown in fig. 14-16, the qPCR melting temperature of each primer for the same candida species showed a stable trend, the N value in the table represents the number of clinical isolates, and two "candida glabrata" in 78 isolates did not match the melting temperature of the four candida species, so only 10 of the 12 collected candida glabrata were confirmed, and thus the three candida species were 100% compared to the identification result of the chromogenic medium, except that the candida glabrata coincidence rate was 83.33%. Two suspected Candida glabrata strains are identified as Candida parapsilosis by DNA sequencing.
Preferably, the candida is cultured for a short time on a plate with a microfiltration membrane, and then transferred to a chromogenic medium, so that colonies are colored by the principle that thiazole blue, which is a chromogenic substance, can react with succinate dehydrogenase in candida to produce blue-purple formazan.
Preferably, the clinical isolates of Candida albicans, Candida tropicalis 21, Candida krusei 15 and Candida glabrata 12 in the step S1 have been identified by Candida chromogenic medium.
Compared with the prior art, the invention has the beneficial effects that: the rapid analysis method of the yeast-type fungi based on membrane culture;
1. the research can not only retain the advantages of a culture method to achieve certain enrichment treatment on the fungus sample, but also can be linked with high-sensitivity qPCR, so that the method only needs to obtain the sufficient fungus quantity detected by the qPCR, and does not need long-time culture;
2. the method adopts a qPCR combined melting curve analysis technology, the qPCR improves the detection sensitivity, simplifies the PCR process, reduces the pollution and the operation complexity caused by the subsequent electrophoresis detection of the conventional PCR, the melting curve analysis after the qPCR can obtain the corresponding characteristic melting temperature according to different sequences of amplification products, and can identify the species source of an amplicon, and three detection targets are designed from three layers of a fungus universal primer, a candida universal primer and a candida specific primer to identify one candida, so the detection specificity is improved;
3. the method comprises the steps of adopting microporous membrane short-time culture, color plate indication, quick genome DNA release of bacterial colonies, real-time quantitative PCR and melting curve analysis to detect the candida, simultaneously evaluating the detection limit and specificity of qPCR, shortening the time of the bacterial colonies visible to naked eyes by more than 50% compared with the conventional culture method by adopting a membrane culture technology, enabling the detection limit of each candida qPCR to be in the range of 1 fg-100 fg and being 1000 times higher than that of the conventional PCR, and displaying the results of the qPCR melting curve analysis of three target points of general fungi, general candida and specific candida, wherein triple amplification can form unique melting temperature combinations aiming at different candida so as to be beneficial to identification of strains, and the whole research mode can be used for quickly and accurately identifying the common candida.
Drawings
FIG. 1 is a schematic structural diagram of the Candida albicans membrane culture result of the present invention;
FIG. 2 is a schematic diagram showing the structure of blue-violet colony spots after the color development of the filter membrane of the present invention;
FIG. 3 is a schematic diagram showing a comparative structure of the filter membrane coloration method and the conventional culture method after 8 hours of culture;
FIG. 4 is a structural diagram showing the comparison of the colony counting results of the filter membrane coloration method and the conventional culture according to the present invention; FIG. 5 is a schematic diagram showing the structure of the colony PCR agarose gel electrophoresis result of the present invention;
FIG. 6 is a diagram showing the qPCR detection melting limit curve map structure of ITS86/ITS4 of the present invention;
FIG. 7 is a diagram showing the qPCR detection melting limit curve map structure of pCps-1F/1R of the present invention;
FIG. 8 is a diagram of a qPCR detection melting limit curve map structure of Caps-1F/1R of the present invention;
FIG. 9 is a schematic diagram of a qPCR detection limit melting curve map structure of CTR1/2 of the present invention;
FIG. 10 is a schematic diagram of a qPCR detection limit melting curve map structure of CGL1/2 of the present invention;
FIG. 11 is a schematic diagram of a qPCR detection limit melting curve map structure of CKRU1/2 according to the present invention;
FIG. 12 is a schematic diagram of a melting curve combination analysis map of Candida albicans subjected to multi-target qPCR;
FIG. 13 is a schematic diagram of a melting curve combination analysis map of Candida tropicalis after multi-target qPCR;
FIG. 14 is a schematic diagram of a melting curve combination analysis map of Candida glabrata after multi-target qPCR according to the present invention;
FIG. 15 is a schematic diagram of a melting curve combination analysis map of Candida krusei after multi-target qPCR;
FIG. 16 is a schematic structural diagram showing the qPCR melting temperature analysis result of the fungus universal primer ITS86/ITS4 for clinical isolates of the present invention;
FIG. 17 is a schematic diagram showing the qPCR melting temperature analysis results of Candida universal primer pCps-1F/1R for clinical isolates of the present invention;
FIG. 18 is a schematic structural diagram of the qPCR melting temperature analysis results of the Candida species specific primers used in the clinical isolates of the present invention.
Detailed Description
Technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a rapid analysis method of yeast-type fungi based on membrane culture comprises the following steps:
s1 test Material
(1) Candida albicans standard strain, Candida tropicalis standard strain, Candida krusei standard strain, Candida glabrata standard strain, 30 Candida albicans clinical isolates, 21 Candida tropicalis clinical isolates, 15 Candida krusei clinical isolates and 12 Candida glabrata clinical isolates.
(2) All clinical isolates used in this experiment have been identified as species using a Candida chromogenic medium.
S2 strain culture
According to different purposes, all strains in the experiment are cultured in a Sapaul solid culture medium (prepared in a laboratory) at 30 ℃ or in a yeast extract peptone glucose (YPD) culture solution (prepared in the laboratory) at 30 ℃ and under 220rpm shaking.
S3 Candida primer selection
Detection primers were selected at different levels from the ribosomal DNA gene coding regions of Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei, namely the 18SrRNA, the transcribed spacer-1, 5.8SrRNA, the transcribed spacer-2 and 28SrRNA five segments, namely the fungal universal primer ITS84/ITS6, the Candida universal primer pCps-1F/1R, the Candida albicans specific primer Caps-1F/1R, the Candida tropicalis specific primer CTR1/2, the Candida glabrata specific primer CGL1/2 and the Candida krusei specific primer CKRU 1/2.
S4, Membrane culture
(1) The culture medium used in the membrane culture technique comprises two parts, a Sapaul plate with a white microporous filter membrane paved on the surface and a chromogenic plate without nutrient components, wherein the cellulose microporous filter membrane allows water and nutrient exchange, but prevents candida cells from passing through, and the chromogenic plate is made of 1% agarose gel containing 0.8mg/ml thiazole blue.
(2) Selecting a Candida albicans colony under aseptic condition, diluting the Candida albicans colony with YPD culture solution 1:1000, respectively taking 10 mu l of diluted bacterial solution, coating an inoculation membrane plate, culturing at 30 ℃, respectively taking out filter membranes at 2h, 4h, 6h, 8h, 12h and 24h, transferring the filter membranes to a color development plate, simultaneously observing the color development condition of the colony on the filter membranes and the growth condition of the colony cultured conventionally, and setting three parallel at each time point.
S5 colony PCR
(1) And (3) selecting the candida tenuis colonies which are subjected to membrane culture and have color development indication, suspending the candida tenuis colonies in 20 mu l of candida lysate, carrying out vortex oscillation, incubating for 5min at 98 ℃ after short vortex oscillation, centrifuging for 10min at 1500Xg, collecting supernatant, diluting with sterile ultrapure water according to a ratio of 1: 5-1: 20, and then respectively using the supernatant as a template to carry out PCR test.
(2) Candida albicans specific primers Casps-1F/1R are selected, the annealing temperature is 58 ℃, sterile ultrapure water is used as a template to serve as a blank control, and the amplification result is analyzed through 2% agarose gel electrophoresis.
S6 extraction of Candida genomic DNA
Respectively taking 1ml of fresh candida bacterial liquid, placing the fresh candida bacterial liquid in a 1.5ml sterile centrifuge tube, centrifuging for 1min at 13400 Xg, discarding supernatant, carrying out wall breaking, column combination, rinsing, elution and other treatments on candida thalli by using a yeast genome DNA extraction kit, finally obtaining a genome sample, quantifying by using an ultraviolet spectrophotometer, and freezing and storing at-30 ℃ for later use.
S7, conventional PCR analysis of primer specificity and detection Limit
(1) And (3) respectively taking 1ng of four candida genomic DNAs, common bacterial genomic DNAs and human genomic DNAs as templates, testing the specificity of the selected 6 pairs of primers according to the PCR parameter setting of the step S5, and taking sterile ultrapure water as a template as a blank control.
(2) Analyzing the amplification result by agarose gel electrophoresis, and respectively carrying out 10-10 ng on 1ng of four candida genomic DNAs6Diluting by times, testing the conventional PCR detection limit of 6 pairs of primers, taking sterile ultrapure water as a template as a blank control, and analyzing the amplification result by agarose gel electrophoresis.
S8 qPCR detection and melting curve analysis of each primer specificity and detection limit
(1) qPCR analysis was performed on the specificity and detection limit of each primer in step S7 using an iQ5qPCR instrument, and 25. mu.l systems were prepared using SYBRGreenPremixExTaq kit, respectively.
(2) The qPCR program was set to 95 ℃ pre-denaturation for 30s, "95 ℃, 5s → 58 ℃, 5s → 72 ℃, 5 s" repeated 35 cycles.
(3) Melting curve analysis was then performed: the temperature was raised from 60 ℃ to 95 ℃ at a rate of 0.1 ℃/s, the change in fluorescence intensity of each qPCR amplicon was recorded, a melting curve was generated, each qPCR threshold was determined by the iQ5 software system default, sterile ultrapure water was used as a template as a blank control, and a set of replicates was set.
S9, qPCR verification of clinical isolate
And (3) carrying out qPCR detection and melting curve analysis on 78 clinical isolates of the candida, and respectively carrying out amplification of a plurality of target points on each candida by using a fungus universal primer, a candida universal primer and a candida specific primer.
S10, data analysis
Mean standard deviation analysis was performed on qPCR data using iQ5 analysis software and microsoft Excel tool.
S11, result
(1) Results of membrane culture
Compared with the conventional culture method, the culture time can be shortened by more than 50%, as shown in figure 1-2, the blue-violet colony spots can be seen by naked eyes after the filter membrane cultured for 8h is developed.
(2) Colony PCR results
The results show that the colony is stained by thiazole blue, the PCR is not affected, except that the PCR of lysate of the colony at a ratio of 1:5 is negative, the templates of the other three dilutions are amplified to form a single band, the size of the product is consistent with the expected result of 272bp, the PCR negative is probably caused by excessively high concentration of the lysate, and the results are shown in FIG. 3:
lane M: DNAmarker;
lane 1: diluting bacterial colony lysate by 1: 5;
lane 2: diluting bacterial colony lysate by 1: 10;
lane 3: diluting bacterial colony lysate at 1: 15;
lane 4: diluting bacterial colony lysate by 1: 20;
lane 5: blank control.
(3) Detection limits and specificities of conventional PCR and qPCR
1. Compared with the detection limit of each primer in the conventional PCR of 1 pg-100 pg, the detection limit of each primer in the qPCR is 1 fg-100 fg, and the overall sensitivity is 1000 times higher than that of the conventional PCR, as shown in FIGS. 4-9 (the abscissa of the qPCR melting curve is temperature (DEG C), and the ordinate is melting peak), the negative derivative (-dRFU/dT) of the relative fluorescence unit data of the amplified gene at each temperature shows a falling trend along with the reduction of the template amount, and the detection limit is the minimum template DNA amount capable of obtaining the melting peak.
2. After the specificity of each primer is analyzed by the electrophoresis of the conventional PCR amplification product, the band specificity is single, but the resolution of agarose electrophoresis is not high, bands with approximate sizes can not be further identified, and qPCR combined melting curve analysis results show that after each candida is identified by three target points qPCR, the triple melting temperatures of the four candida species are 86.5-85.5 ℃ of white, 74.5-85 ℃ of tropical, 74.5-85 ℃ of smooth, 74.5-84.5-75 ℃ of smooth, 75-86.5-90 ℃ of krusei respectively according to the sequences of the fungal universal-candida specific primers, and the results are shown in fig. 10-13 (in the figures, A-D are the triple melting peaks of candida albicans, candida tropicalis, candida glabrata and candida krusei respectively).
(4) Clinical isolates of Candida qPCR analysis
As shown in fig. 14-16, the qPCR melting temperature of each primer for the same candida species showed a stable trend, the N value in the table represents the number of clinical isolates, and two "candida glabrata" in 78 isolates did not match the melting temperature of the four candida species, so only 10 of the 12 collected candida glabrata were confirmed, and thus the three candida species were 100% compared to the identification result of the chromogenic medium, except that the candida glabrata coincidence rate was 83.33%. Two suspected Candida glabrata strains are identified as Candida parapsilosis by DNA sequencing.
After candida is cultured for a short time on a flat plate paved with a microporous filter membrane, the candida is transferred into a chromogenic culture medium, a colony is colored by the principle that a chromogenic substance, namely thiazole blue, can react with succinate dehydrogenase in the candida to generate blue-purple formazan, so that the sensitivity of macroscopic observation is improved, qPCR analysis is immediately transferred, and the overall detection time is shortened;
in step S1, Candida albicans clinical isolates, Candida tropicalis clinical isolates, Candida krusei clinical isolates, 15 Candida krusei clinical isolates and 12 Candida glabrata clinical isolates all adopt Candida chromogenic medium to identify species.
Those not described in detail in this specification are within the skill of the art.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A rapid analysis method of yeast-type fungi based on membrane culture is characterized in that: the method comprises the following steps:
s1 test Material
(1) Candida albicans standard strain, Candida tropicalis standard strain, Candida krusei standard strain, Candida glabrata standard strain, 30 Candida albicans clinical isolates, 21 Candida tropicalis clinical isolates, 15 Candida krusei clinical isolates and 12 Candida glabrata clinical isolates.
(2) All clinical isolates used in this experiment have been identified by using a CHROMagar (Colomajia) chromogenic medium dedicated to clinical Candida culture.
S2 strain culture
According to different purposes, all strains in the experiment are cultured in a Sapaul solid culture medium (prepared in a laboratory) at 30 ℃ or in a yeast extract peptone glucose (YPD) culture solution (prepared in the laboratory) at 30 ℃ and under 220rpm shaking.
S3 Candida primer selection
Detection primers were selected at different levels from the ribosomal DNA gene coding regions of Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei, namely the 18SrRNA, the transcribed spacer-1, 5.8SrRNA, the transcribed spacer-2 and 28SrRNA five segments, namely the fungal universal primer ITS84/ITS6, the Candida universal primer pCps-1F/1R, the Candida albicans specific primer Caps-1F/1R, the Candida tropicalis specific primer CTR1/2, the Candida glabrata specific primer CGL1/2 and the Candida krusei specific primer CKRU 1/2.
S4, Membrane culture
(1) The culture medium used by the membrane culture technology comprises two parts, namely a Sapaul plate and a color development plate, wherein the surfaces of the Sapaul plate are paved with sterile white cellulose microporous filter membranes, and the color development plate does not contain nutrient components, and the cellulose microporous filter membranes allow water and nutrient to be exchanged but prevent candida cells from passing through; in addition, a thin layer of Sapaul solid medium with a thickness of 2mm was attached to the microfiltration membrane. The color development plate is made of 1% agarose gel containing 0.5-1.0 mg/ml thiazole blue. A conventional culture experiment group using saprola plates was set up in parallel with the membrane culture experiment.
(2) Selecting a Candida albicans colony under aseptic condition, diluting the Candida albicans colony with YPD culture solution 1:1000, respectively taking 10 mu l of diluted bacterial solution, coating an inoculation membrane plate, culturing at 30 ℃, respectively taking out filter membranes at 2h, 4h, 6h, 8h, 12h and 24h, transferring the filter membranes to a color development plate, simultaneously observing the color development condition of the colony on the filter membranes and the growth condition of the colony cultured conventionally, and setting three parallel at each time point.
S5 colony PCR
(1) And (3) selecting the candida tenuis colonies which are subjected to membrane culture and have color development indication, suspending the candida tenuis colonies in 20 mu l of candida lysate, carrying out vortex oscillation, incubating for 5min at 98 ℃ after short vortex oscillation, centrifuging for 10min at 1500Xg, collecting supernatant, diluting with sterile ultrapure water according to a ratio of 1: 5-1: 20, and then respectively using the supernatant as a template to carry out PCR test.
(2) Candida albicans specific primers Casps-1F/1R are selected, the annealing temperature is 58 ℃, sterile ultrapure water is used as a template to serve as a blank control, and the amplification result is analyzed through 2% agarose gel electrophoresis.
S6 extraction of Candida genomic DNA
Respectively taking 1ml of fresh candida bacterial liquid, placing the fresh candida bacterial liquid in a 1.5ml sterile centrifuge tube, centrifuging for 1min at 13400 Xg, discarding supernatant, carrying out wall breaking, column combination, rinsing, elution and other treatments on candida thalli by using a yeast genome DNA extraction kit, finally obtaining a genome sample, quantifying by using an ultraviolet spectrophotometer, and freezing and storing at-30 ℃ for later use.
S7, conventional PCR analysis of primer specificity and detection Limit
(1) And (3) respectively taking 1ng of four candida genomic DNAs, common bacterial genomic DNAs and human genomic DNAs as templates, testing the specificity of the selected 6 pairs of primers according to the PCR parameter setting of the step S5, and taking sterile ultrapure water as a template as a blank control.
(2) Analyzing the amplification result by agarose gel electrophoresis, and respectively carrying out 10-10 ng on 1ng of four candida genomic DNAs6Diluting by times, testing the conventional PCR detection limit of 6 pairs of primers, taking sterile ultrapure water as a template as a blank control, and analyzing the amplification result by agarose gel electrophoresis.
S8 qPCR detection and melting curve analysis of each primer specificity and detection limit
(1) qPCR analysis was performed on the specificity and detection limit of each primer in step S7 using an iQ5qPCR instrument, and 25. mu.l systems were prepared using SYBRGreen Premix ExTaq kit, respectively.
(2) The qPCR program was set to 95 ℃ pre-denaturation for 30s, "95 ℃, 5s → 58 ℃, 5s → 72 ℃, 5 s" repeated 35 cycles.
(3) Melting curve analysis was then performed: the temperature was raised from 60 ℃ to 95 ℃ at a rate of 0.1 ℃/s, the change in fluorescence intensity of each qPCR amplicon was recorded, a melting curve was generated, each qPCR threshold was determined by the iQ5 software system default, sterile ultrapure water was used as a template as a blank control, and a set of replicates was set.
S9, qPCR verification of clinical isolate
And (3) carrying out qPCR detection and melting curve analysis on 78 clinical isolates of the candida, and respectively carrying out amplification of a plurality of target points on each candida by using a fungus universal primer, a candida universal primer and a candida specific primer.
S10, data analysis
Mean standard deviation analysis was performed on qPCR data using iQ5 analysis software and microsoft Excel tool.
S11, result
(1) Results of membrane culture
Compared with the conventional culture method, the culture time can be shortened by more than 50%, as shown in the figure 1-4, blue-purple colony spots can be seen by naked eyes after the filter membrane cultured for 8h is developed. The bacterial colony counting can be carried out after the membrane chromogenic culture is carried out for 8h, the counting result is consistent with the counting result of the bacterial colony cultured for 12h, and meanwhile, the counting result is also consistent with the counting result of the traditional Sabouraud's dextrose agar culture. But the time at which colonies of the former can be counted with the naked eye is significantly advanced.
(2) Colony PCR results
The results show that the colony is stained by thiazole blue, the PCR is not affected, except that the PCR of lysate of the colony at a ratio of 1:5 is negative, the templates of the other three dilutions all amplify a single band, the size of the product is consistent with the expected result of 272bp, the PCR negative is probably caused by excessively high concentration of the lysate, and the results are shown in FIG. 5:
lane M: DNAmarker;
lane 1: diluting bacterial colony lysate by 1: 5;
lane 2: diluting bacterial colony lysate by 1: 10;
lane 3: diluting bacterial colony lysate at 1: 15;
lane 4: diluting bacterial colony lysate by 1: 20;
lane 5: blank control.
(3) Detection limits and specificities of conventional PCR and qPCR
1. Compared with the detection limit of each primer in the conventional PCR of 1 pg-100 pg, the detection limit of each primer in the qPCR is 1 fg-100 fg, and the overall sensitivity is 1000 times higher than that of the conventional PCR, as shown in FIGS. 6-11 (the abscissa of the qPCR melting curve is temperature (. degree. C.) and the ordinate is melting peak), the negative derivative (-dRFU/dT) of the relative fluorescence unit data of the amplified gene at each temperature shows a falling trend as the amount of the template decreases, and the detection limit is the minimum amount of template DNA for obtaining the melting peak.
2. After the specificity of each primer is analyzed by the electrophoresis of the conventional PCR amplification product, the band specificity is single, but the resolution of agarose electrophoresis is not high, bands with approximate sizes can not be further identified, and qPCR combined melting curve analysis results show that after each candida is identified by three target points qPCR, the triple melting temperatures of the four candida species are 86.5-85.5 ℃ of white, 74.5-85 ℃ of tropical, 74.5-85 ℃ of smooth, 74.5-84.5-75 ℃ of smooth, 75-86.5-90 ℃ of krusei respectively according to the sequences of the fungal universal-candida specific primers, and the results are shown in fig. 12-15 (in the figures, A-D are the triple melting peaks of candida albicans, candida tropicalis, candida glabrata and candida krusei respectively).
(4) Clinical isolates of Candida qPCR analysis
As shown in fig. 16-18, the qPCR melting temperature of each primer for the same candida species showed a stable trend, the N value in the table represents the number of clinical isolates, and two of 78 isolates had "candida glabrata" that did not match the melting temperature of the four candida species, so only 10 of 12 collected candida glabrata identified by the chromogenic medium were identified, and thus compared with the identification result of the chromogenic medium, all three candida species were 100% except for the candida glabrata coincidence rate of 83.33%. Two suspected Candida glabrata strains are identified as Candida parapsilosis by DNA sequencing.
2. The rapid analysis method of yeast-type fungi based on membrane culture according to claim 1, characterized in that: the candida is transferred into a chromogenic culture medium after being cultured for a short time on a plate paved with a millipore filter membrane, and colonies are colored by the principle that blue thiazole which is a chromogenic substance can react with succinate dehydrogenase in the candida to generate blue-purple formazan.
3. The rapid analysis method of yeast-type fungi based on membrane culture according to claim 1, characterized in that: the clinical isolates of 30 candida albicans, 21 candida tropicalis, 15 candida krusei and 12 candida glabrata in the step S1 adopt special candida chromogenic culture medium to identify the species.
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